A Method for Producing An Active Hepatocyte Growth Factor (HGF)

ABSTRACT

The present invention provides a method for producing active hepatocyte growth factor activator (HGFA) and active hepatocyte growth factor (HGF) without using animal serum. The present invention relates to a method for producing active HGFA without using animal serum. The method is characterized in that it comprises a step of obtaining a culture supernatant comprising pro-HGFA by culturing mammalian cells expressing inactive hepatocyte growth factor activator (pro-HGFA) in a medium without serum, and a step of adjusting the culture supernatant comprising pro-HGFA obtained in the above step to weakly acidic to convert pro-HGFA into active HGFA. The present invention also relates to a method for producing active HGF with HGFA produced by said method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/078,568, filed on Aug. 21, 2018, which is the National Stage ofInternational Application No. PCT/JP2017/010355, filed on Mar. 15, 2017and claims the benefit of Japanese Application No. 2016-054128, filed onMar. 17, 2016. The disclosures of the prior applications is incorporatedherein by reference.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an ASCII text file named SEQLIST1.txt. The ASCII textfile, created on Mar. 9, 2017, is 11,671 bytes in size. The material inthe ASCII text file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a method for producing activehepatocyte growth factor activator (also referred to herein as “activeHGFA”) and active hepatocyte growth factor (also referred to herein as“active HGF”) without using animal serum.

BACKGROUND ART

HGF is a factor having hepatic parenchymal cell proliferation activitythat is purified from the blood plasma of human fulminant hepatitispatients (Patent Literature 1 and Non-Patent Literature 1), and has beenreported as having various pharmacological effects such as antitumoraleffect, enhancement of cell-mediated immunity, wound therapeutic effect,and tissue regeneration promotional effect (Patent Literature 2).

Until now, the gene encoding the aforementioned HGF has been cloned andproduced by recombinant DNA technology (Patent Literatures 3-5).Moreover, it is known that HGF takes single-stranded and double-strandedforms which are composed of 2 types of subunits (a chain ofapproximately 60 kDa and (3 chain of approximately 30 kDa), where thesingle-stranded form does not have bioactivity and gains bioactivity inthe double-stranded form. Further, it is known that in the production byrecombinant DNA technology, HGF can be obtained as the activedouble-stranded form under culturing with animal serum, but underculturing without animal serum, the majority of the HGF produced isobtained as the inactive single-stranded form (e.g. Patent Literature6). Since a protease contained in animal serum is involved in theconversion from the single-stranded inactive hepatocyte growth factorform (also referred to herein as “pro-HGF”) into the double-strandedactive HGF form, it is thought necessary to use animal serum in order toefficiently obtain active HGF.

On the other hand, in recent years, the mainstream in the production ofbiological material by recombinant DNA technology is culturing withoutanimal serum in order to avoid the risk of virus contamination etc.Accordingly, in order to manufacture active HGF with a medium withoutanimal serum, it is necessary to convert the single-stranded pro-HGFform into active HGF by some means. HGFA that can convert pro-HGF intoactive HGF (Patent Literature 7), or serine protease such as urokinaseplasminogen activator (Non-Patent Literature 2) are known as such means.However, there are problems that these enzymes that can convert pro-HGFinto active HGF are serum-derived, and when they are required to produceby integration of the gene into microorganisms or animal cells forproduction, they are produced as the precursor forms in a serum-freeculture and therefore difficult to use as they are.

CITATION LIST Patent Literatures

[Patent Literature 1] Japanese Published Unexamined Patent ApplicationPublication No. S63-22526

[Patent Literature 2] Japanese Patent No. 2747979

[Patent Literature 3] Japanese Patent No. 2577091

[Patent Literature 4] Japanese Patent No. 2859577

[Patent Literature 5] Japanese Patent No. 3072628

[Patent Literature 6] Japanese Patent No. 3213985

[Patent Literature 7] Japanese Published Unexamined Patent ApplicationPublication No. H5-103670

Non-Patent Literatures

[Non-Patent Literature 1] J. Clin. Invest., 81, 414(1988)

[Non-Patent Literature 2] JGH 26(2011) Suppl. 1; 188-202, p. 192

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The object of the present invention is to provide a method for producingactive HGFA and active HGF without using animal serum.

The another object of the present invention is to provide active HGFA,active HGF, and preparations thereof produced by the method of thepresent invention.

Means for Solving the Problems

As a result of extensive investigation by the present inventors to solvethe above problems, it was found that by culturing mammalian cellsexpressing inactive hepatocyte growth factor activator (pro-HGFA) in amedium without serum to obtain the culture supernatant thereof, andsubjecting the aforementioned culture supernatant to a particulartreatment, pro-HGFA contained in the aforementioned culture supernatantcan be converted into active HGFA. Accordingly, since pro-HGF producedunder culturing without animal serum can be converted into active HGF byactive HGFA similarly produced under culturing without animal serum,active HGF that does not contain animal serum-derived component or apreparation comprising the same can be produced.

Accordingly, the present invention encompasses the following aspects:

[1] A method for producing active hepatocyte growth factor activator(HGFA), characterized in that it comprises: Step 1:

a step of obtaining a culture supernatant comprising pro-HGFA byculturing mammalian cells expressing inactive hepatocyte growth factoractivator (pro-HGFA) in a medium without serum, and Step 2:

a step of adjusting the culture supernatant comprising pro-HGFA obtainedin the above step to weakly acidic to convert pro-HGFA into active HGFA.

[2] The production method according to [1], characterized in that saidstep further comprises adding sulfated polysaccharides to said culturesupernatant.

[3] The production method according to [1] or [2], characterized in thatsaid step of adjusting the culture supernatant to weakly acidic is astep of adjusting pH to 4.0-6.0.

[4] The production method according to any of [1] to [3], characterizedin that said step of adjusting the culture supernatant to weakly acidicis performed at a temperature of 15-40° C.

[5] The production method according to any of [1] to [4], characterizedin that said culture supernatant is obtained after a decline in thesurvival rate of mammalian cells in culture.

[6] The production method according to any of [1] to [5], characterizedin that said mammalian cell is a Chinese hamster ovary (CHO) cell.

[7] The production method according to any of [1] to [6], characterizedin that said pro-HGFA has the amino acid sequence shown in SEQ ID NO. 2.

[8] The production method according to any of [1] to [7], characterizedin that said culture supernatant is said culture supernatant per se, adilution of said culture supernatant, a concentrate of said culturesupernatant, or a partially purified product of said culturesupernatant.

[9] Active HGFA characterized in that it is obtained by the productionmethod according to any of [1] to [8].

[10] A method for production active hepatocyte growth factor (HGF),characterized in that it comprises a step of allowing active HGFA to acton a culture supernatant comprising inactive hepatocyte growth factor(pro-HGF) to convert said pro-HGF into active HGF,

wherein

said culture supernatant comprising pro-HGF is a culture supernatantobtained by culturing cells expressing pro-HGF in a medium withoutserum, and

said active HGFA is produced by the method according to any of [1] to[8].

[11] The production method according to [10], characterized in that saidmedium for culturing cells expressing pro-HGF is a medium without anyanimal-derived components.

[12] The production method according to [10] or [11], characterized inthat said pro-HGF has the amino acid sequence shown in SEQ ID NO. 1.

[13] Active HGF characterized in that it is obtained by the productionmethod according to any of [10] to [12].

Those skilled in the art shall recognize that an invention of anycombination of one or more characteristics of the present inventiondescribed above is also encompassed by the scope of the presentinvention.

Effects of the Invention

According to the present invention, a method for producing active HGFAand active HGF without using animal serum is provided.

In the method for producing the active HGF of the present invention,since there is no need to use any animal serum in the production processthereof, a composition comprising the active HGF obtained by theaforementioned production method does not comprise animal serum-derivedcomponent and can be extremely safely applied to human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that an HGFA culture supernatant prepared by activation ofa pro-HGFA culture supernatant activates pro-HGF in a culturesupernatant derived from CHO cells comprising pro-HGF.

FIG. 2 shows the verification result employing design of experiments(DoE) regarding conditions under which the pro-HGFA culture supernatantis activated.

FIG. 3 shows SDS-PAGE after chromatographic purification that employs amultimodal anion exchanger Capto Adhere as the chromatography supportand 20 mM Tris-hydrochloride buffer (pH 8.0) comprising 0.25 M arginineand 0.7 M arginine as the eluent.

FIG. 4 shows SDS-PAGE after chromatographic purification that employs amultimodal anion exchanger Capto Adhere as the chromatography supportand 20 mM Tris-hydrochloride buffer (pH 8.0) comprising 1 M arginine asthe eluent.

FIG. 5 shows non-reductive and reductive SDS-PAGE results of thepurified product at each stage of purification where purificationsimilar to Example 5 was performed with a culture supernatant comprisingpro-HGF that is not activated by a HGFA culture supernatant.

FIG. 6 shows the result of measuring cell proliferation activity in thepresence of TGFβ for purified HGF obtained in Example 5.

DESCRIPTION OF EMBODIMENTS

Reference herein to “active hepatocyte growth factor (active HGF),”unless otherwise explicitly shown, is construed as referring to thedouble-stranded activated HGF form, and is used in discrimination withinactive hepatocyte growth factor (pro-HGF) which is the single-strandedinactive form thereof.

In the present invention, HGF may comprise HGF derived from humans,mice, rats, rabbits, or other animals. In the present invention, HGF ispreferably HGF derived from humans.

In the present invention, human HGF (hHGF) includes a polypeptide havingthe amino acid sequence shown in SEQ ID NO. 1 or a variant thereof. Avariant of the polypeptide having the amino acid sequence shown in SEQID NO. 1 includes a polypeptide having an amino acid sequence havingaddition, deletion, or substitution of one or multiple amino acids tothe amino acid sequence shown in SEQ ID NO. 1, as well as having HGFactivity similar to or more than the polypeptide having the amino acidsequence shown in SEQ ID NO. 1 or which may be activated to have theactivity. “Multiple” as used herein is 2-150, more preferably 2-80, morepreferably 2-70, more preferably 2-60, more preferably 2-50, morepreferably 2-40, more preferably 2-30, more preferably 2-20, morepreferably 2-10, or more preferably 2-5.

A variant of the polypeptide having the amino acid sequence shown in SEQID NO. 1 also includes a polypeptide having an amino acid sequenceshowing at least 80%, more preferably at least 85%, and more preferablyat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with the amino acid sequence shown in SEQ ID NO. 1, as well ashaving HGF activity similar to or more than the polypeptide having theamino acid sequence shown in SEQ ID NO. 1 or which may be activated tohave the activity.

A variant of the polypeptide having the amino acid sequence shown in SEQID NO. 1 also includes a polypeptide having the amino acid sequenceencoded by a polynucleotide that hybridizes under stringent condition toa polynucleotide encoding the amino acid sequence shown in SEQ ID NO. 1,as well as having HGF activity similar to or more than the polypeptidehaving the amino acid sequence shown in SEQ ID NO. 1 or which may beactivated to have the activity.

In the present invention, a “stringent condition” can include thosewhere in the post hybridization washing, hybridization is achieved withwashing at for example a condition of “2×SSC, 0.1% SDS, 50° C.,” acondition of “2×SSC, 0.1% SDS, 42° C.,” or a condition of “1×SSC, 0.1%SDS, 37° C.,” and a more stringent condition can include those wherehybridization is achieved with washing at for example conditions of“2×SSC, 0.1% SDS, 65° C.,” “0.5×SSC, 0.1% SDS, 42° C.,” “0.2×SSC, 0.1%SDS, 65° C.,” or “0.1×SSC, 0.1% SDS, 65° C.” (1×SSC is 150 mM sodiumchloride, 15 mM sodium citrate, pH 7.0). More particularly, as a methodthat employs Rapid-hyb buffer (Amersham Life Science), it is conceivableto perform prehybridization at 68° C. for 30 minutes or more, afterwhich a probe is added and retained at 68° C. for 1 hour or more toallow formation of hybrids, and then to perform three washes in 2×SSCand 0.1% SDS at room temperature for 20 minutes, three washes in 1×SSCand 0.1% SDS at 37° C. for 20 minutes, and finally two washes in 1×SSCand 0.1% SDS at 50° C. for 20 minutes. More preferably, using asolution, for example, comprising 5×SSC, 7% (W/V) SDS, 100 μg/mLdenatured salmon sperm DNA, and 5×Denhardt's solution (1×Denhardt'ssolution comprises 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin,and 0.2% Ficoll) as prehybridization and hybridization solutions,prehybridization is performed at 65° C. for 30 minutes to 1 hour andhybridization is performed at the same temperature overnight (6-8hours). In addition, it is also possible to perform for exampleprehybridization in Expresshyb Hybridization Solution (CLONTECH) at 55°C. for 30 minutes or more, add a labeled probe and incubate at 37-55° C.for 1 hour or more, and three washes in 2×SSC and 0.1% SDS at roomtemperature for 20 minutes and then one washing in 1×SSC and 0.1% SDS at37° C. for 20 minutes. Here, a more stringent condition can be achievedfor example by raising the temperature for prehybridization,hybridization, or second washing. For example, the temperature forprehybridization and hybridization can be 60° C., or 65° C. or 68° C.for a further stringent condition. Those skilled in the art will be ableto set conditions for obtaining isoforms, allelic variants, andcorresponding genes derived from other organism species for the gene ofthe present invention by factoring in various conditions such as otherprobe concentration, probe length, and reaction time in addition toconditions such as salt concentration of such a buffer and temperature.For a detailed protocol of the hybridization method, reference can bemade to “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold SpringHarbor Press (1989); in particular Section 9.47-9.58), “CurrentProtocols in Molecular Biology” (John Wiley & Sons (1987-1997); inparticular Section 6.3-6.4), “DNA Cloning 1: Core Techniques, APractical Approach 2nd ed.” (Oxford University (1995); in particularSection 2.10 for conditions), and the like.

Reference herein to “active hepatocyte growth factor activator (activeHGFA),” unless otherwise explicitly shown, is construed as referring toactivated HGFA, and is used in discrimination with inactive hepatocytegrowth factor activator (pro-HGFA) which is the inactive form thereof.

In the present invention, HGFA may include HGFA derived from human,mouse, rat, rabbit, or other animals. In the present invention, HGFA ispreferably HGFA derived from humans.

In the present invention, human HGFA includes a polypeptide having anamino acid sequence shown in SEQ ID NO. 2 or a variant thereof. Thevariant of the polypeptide having the amino acid sequence shown in SEQID NO. 2 includes a polypeptide having an amino acid sequence havingaddition, deletion, or substitution of one or multiple amino acids tothe amino acid sequence shown in SEQ ID NO. 2, as well as having HGFactivity similar to or more than the polypeptide having the amino acidsequence shown in SEQ ID NO. 2 or which may be activated to have theactivity. “Multiple” as used herein is 2-150, more preferably 2-80, morepreferably 2-70, more preferably 2-60, more preferably 2-50, morepreferably 2-40, more preferably 2-30, more preferably 2-20, morepreferably 2-10, or more preferably 2-5.

The variant of the polypeptide having the amino acid sequence shown inSEQ ID NO. 2 also includes a polypeptide having an amino acid sequenceshowing at least 80%, more preferably at least 85%, and more preferablyat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with the amino acid sequence shown in SEQ ID NO. 2, as well ashaving HGFA activity similar to or more than the polypeptide having theamino acid sequence shown in SEQ ID NO. 2 or which may be activated tohave the activity.

The variant of the polypeptide having the amino acid sequence shown inSEQ ID NO. 2 also includes a polypeptide having the amino acid sequenceencoded by a polynucleotide that hybridizes under stringent condition toa polynucleotide encoding the amino acid sequence shown in SEQ ID NO. 2,as well as having HGF activity similar to or more than the polypeptidehaving the amino acid sequence shown in SEQ ID NO. 2 or which may beactivated to have the activity.

The present invention is described in detail below.

In one aspect, the present invention relates to a method for producingactive HGFA without using animal serum. Specifically, the method forproducing the active HGFA of the present invention is a method forproducing active HGFA by subjecting a culture supernatant comprisingpro-HGFA recombinantly expressed in mammalian cells to a given treatmentto thereby allow conversion into active HGFA. Since animal serum is notused in the conversion into active HGFA, according to the presentmethod, the pro-HGFA obtained or a composition comprising the same hassignificantly lower possibility of inviting the risk of beingcontaminated with infective materials such as virus derived from cellsof other animal species or other individuals, and can be employed forvarious purposes as a highly safe biological material.

Specifically, in one embodiment, the method for producing the activeHGFA of the present invention is characterized in that it comprises thefollowing steps: Step 1:

a step of obtaining a culture supernatant comprising pro-HGFA byculturing mammalian cells expressing pro-HGFA in a medium without serum,and Step 2:

a step of adjusting the culture supernatant comprising pro-HGFA obtainedin the above step to weakly acidic to convert pro-HGFA into active HGFA.

In another embodiment, the HGFA production method of the presentinvention is characterized in that it comprises a step of adjusting theculture supernatant comprising pro-HGFA to weakly acidic to convertpro-HGFA into active HGFA, wherein said culture supernatant is a culturesupernatant obtained by culturing mammalian cells expressing pro-HGFA ina medium without serum.

Weak acidification of the culture supernatant is a treatment forconverting pro-HGFA into active HGFA. Since conversion from pro-HGFAinto active HGFA occurs by weak acidification alone without externallyadding enzymes etc. to the culture supernatant, mammalian cell-derivedcomponents are thought to be involved in the conversion from pro-HGFAinto active HGFA, and weak acidification is a means for activating saidmammalian cell-derived components. Weak acidification may be performedby means well-known to those skilled in the art, such as adding forexample an acidic solution (inorganic acids such as hydrochloric acid,sulfuric acid, and phosphoric acid, or organic acids such as aceticacid, succinic acid, and citric acid) at an appropriate concentration.In one embodiment of the present invention, “weakly acidic” is a rangeof pH 4.0-6.0, preferably 5.0-6.0, and more preferably 5.3-5.6, forexample pH 5.5.

In the present invention, a “culture supernatant comprising pro-HGFA”(also referred to herein as a “pro-HGFA culture supernatant”) is afraction comprising pro-HGFA that is obtained by cell culturingmammalian cells expressing pro-HGFA, which can be obtained from a cellculture of said mammalian cells by those skilled in the art according toconventional means. For example, the pro-HGFA culture supernatant may bea fraction where residues are removed from a cell culture of saidmammalian cells by a means such as centrifugation.

In the present invention, the culture supernatant may be any fractionprepared by applying any treatment to the culture supernatant to anextent that the biological activity of pro-HGFA is not lost.Accordingly, in the present invention, the culture supernatant includes,but is not limited to, the culture supernatant per se, and a dilution, aconcentrate or a partially purified product of the culture supernatant.

In a preferred embodiment of the present invention, the “step ofconverting pro-HGFA into active HGFA” further comprises adding sulfatedpolysaccharides to said culture supernatant. By adding sulfatedpolysaccharides, the conversion from pro-HGFA into active HGFA can beperformed more efficiently. The timing for adding sulfatedpolysaccharides may be at any time point of before weak acidification ofsaid culture supernatant, simultaneously with weak acidification, orafter weak acidification. Moreover, the amount of sulfatedpolysaccharides added may vary depending on e.g. the type of sulfatedpolysaccharides used, and may be added at an amount of 0.01-50 mg, morepreferably 0.1-20 mg, for example 1 mg per 1 mL of said pro-HGFA culturesupernatant.

Sulfated polysaccharides that can be used for the method for producingthe active HGFA of the present invention can include, but are notlimited to, peparin, dextran sulfate, chondroitin sulfate, fucoidan, andsalts thereof. In a preferred embodiment of the present invention,dextran sulfate is used.

In a preferred embodiment of the present invention, the “step ofconverting pro-HGFA into active HGFA” is performed at a temperature of15-40° C., preferably 20-37° C., for example 25° C. By employing saidtemperature range, the conversion of pro-HGFA into active HGFA can bemade more efficient.

In the method for producing the active HGFA of the present invention,the “step of converting pro-HGFA into active HGFA” is performed for alength of time sufficient to recognize the desired HGFA activity afterweak acidification. Such a length of time may vary depending on pH, thepresence or absence of sulfated polysaccharide used in combination, andtemperature condition etc., and can be 1-15 hours, for example 6-8 hoursafter weak acidification.

In one embodiment of the present invention, the pro-HGFA culturesupernatant is a culture supernatant that is obtained after a decline inthe survival rate of mammalian cells in culture. Along with the declinein the survival rate of the mammalian cells, the animal cell-derivedcomponents involved in the conversion from pro-HGFA into active HGFA areeluted out from dead cells, and can be sufficiently collected in thepro-HGFA culture supernatant. The “decline in the survival rate ofmammalian cells in culture” herein refers to the decline in the survivalrate of the mammalian cells after proliferation to maximum cell density.In the present invention, the survival rate of mammalian cells in thepro-HGFA culture supernatant is preferably 95% or less, more preferably80% or less, for example 70%.

Since an enzyme derived from host cell lysosome is thought to beinvolved in the activation from pro-HGFA to active HGFA, an enzymederived from lysosome may be added to apply treatment.

Mammalian cells that can be used in the method for producing the activeHGFA of the present invention can include, but are not limited to,Chinese hamster ovary (CHO) cells, HeLa cells, HEK cells (including HEK293 cells), COS cells, NSO mouse myeloma cells, Sp2/0 mouse myelomacells, and the like. In a preferred embodiment of the present invention,CHO cells are used as mammalian cells expressing pro-HGFA.

The present invention also relates to a composition comprising activeHGFA or active HGFA produced by the method for producing the active HGFAof the present invention. Since the composition comprising active HGFAor active HGFA of the present invention can be produced without usinganimal serum from recombinantly expressed pro-HGFA without using animalserum, it can be used as a highly safe biological material e.g. for themethod for producing the active HGF described below.

In another aspect, the present invention relates to a method forproducing active HGF. The method for producing the active HGF of thepresent invention comprises allowing active HGFA obtained by the methodfor producing the active HGFA of the present invention to act on aculture supernatant comprising pro-HGF recombinantly expressed in amedium similarly without serum to convert pro-HGF into active HGF.According to this method, since active HGF can be produced withoutemploying animal serum in all of the steps including obtaining activeHGFA employed for conversion into active HGF, the active HGF obtained ora composition comprising the same can be used as a highly safepharmaceutical material that eliminates the risk of being contaminatedwith infective materials such as virus.

Specifically, in one embodiment, the method for producing the active HGFof the present invention is characterized in that it comprises a step ofallowing active HGFA to act on a culture supernatant comprising pro-HGFto convert said pro-HGF into active HGF,

wherein

said culture supernatant comprising pro-HGF is a culture supernatantobtained by culturing cells expressing pro-HGF in a medium withoutserum, and

said active HGFA is produced by the above method for producing theactive HGFA of the present invention.

Moreover, in another embodiment, the method for producing the active HGFof the present invention is characterized in that it comprises thefollowing steps: Step A:

a step of adjusting the culture supernatant comprising pro-HGFA toweakly acidic to convert pro-HGFA into active HGFA, wherein said culturesupernatant is a culture supernatant obtained by culturing mammaliancells expressing pro-HGFA in a medium without serum, Step B:

a step of obtaining a culture supernatant comprising pro-HGF byculturing cells expressing pro-HGF in a medium without serum, Step C:

a step of allowing the active HGFA obtained in said step A to act on theculture supernatant comprising pro-HGF obtained in said step B toconvert said pro-HGF into active HGF.

In the present invention, a “culture supernatant comprising pro-HGF” isa fraction comprising pro-HGF that is obtained by culturing cellsexpressing pro-HGF, and those skilled in the art can obtain the samefrom a culture of said cells according to conventional means. Forexample, a culture supernatant comprising pro-HGF may be a fractionwhere residues are removed from a culture of said cells by a means suchas centrifugation.

In the method for producing the active HGF of the present invention, aculture supernatant obtained by culturing mammalian cells expressingpro-HGFA in a medium without serum may be employed as it is, or adilution, a concentrate, or a partially or completely purified productof the aforementioned culture supernatant may be employed as the activeHGFA that is allowed to act on the “culture supernatant comprisingpro-HGF.”

In the present invention, a mammalian cell expressing pro-HGFA can beobtained by, but not limited to, creating a vector comprising a nucleicacid encoding pro-HGFA, and introducing this into a host cell mammaliancell to allow transformation. Similarly, a cell expressing pro-HGF canbe obtained by creating a vector comprising a nucleic acid encodingpro-HGF, and introducing this into a host cell to allow transformation.

A gene expression vector etc. can be used as the above-described vector.A “gene expression vector” is a vector which has the function to expressthe base sequence that the nucleic acid of interest has, and may includea promoter sequence, an enhancer sequence, a repressor sequence, aninsulator sequence, and the like for controlling the expression of saidbase sequence. These sequences are not particularly limited as long asthey function in the host cell.

The means to create the vector comprising the nucleic acid of interestis well-known to those skilled in the art, and those skilled in the artcan suitably select an appropriate method. For example, such a means caninclude, but is not limited to, a ligase reaction that utilizes arestriction enzyme site and the like (Current Protocols in MolecularBiology, John Wiley & Sons (1987) Section 11.4-11.11; Molecular Cloning,A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1989) Section5.61-5.63).

The cells expressing pro-HGF are not particularly limited as long asthey can express pro-HGF, and include, for example, insect cells,eukaryotic cells, mammalian cells. Preferably, in terms of efficientlyexpressing a nucleic acid encoding pro-HGF derived from human, mammaliancells, e.g., CHO cells, HEK cells (including HEK 293 cells), HeLa cells,NSO cells, or SP2/0 mouse myeloma cells are used. In a preferredembodiment of the invention, CHO cells are used as the mammalian cellexpressing pro-HGF.

The means for introducing the above vector into a host cell iswell-known, and those skilled in the art can suitably select anappropriate method. Examples can include, but are not limited to, forintroduction of a vector into a host cell, electroporation method (Chuet al. (1987) Nucleic Acids Res. 15: 1311-26), cationic liposome method,electrical pulse perforation method (Current Protocols in MolecularBiology, John Wiley & Sons (1987) Section 9.1-9.9), direct inject methodusing a capillary glass tube, microinjection method, lipofection(Derijard (1994) Cell 7: 1025-37; Lamb (1993) Nature Genetics 5: 22-30;Rabindran et al. (1993) Science 259: 230-4), lipofectamine method(Thermo Fisher Scientific), calcium phosphate method (Chen and Okayama(1987) Mol. Cell. Biol. 7: 2745-52), DEAE dextran method (Lopata et al.(1984) Nucleic Acids Res. 12: 5707-17; Sussman and Milman (1985) Mol.Cell. Biol. 4: 1642-3), FreeStyle MAX Reagent (Thermo FisherScientific), and the like.

In regards to the serum-free medium used for culturing cells expressingpro-HGFA and cells expressing pro-HGF, those skilled in the art cansuitably select an appropriate composition depending on the type of hostcell etc. used. Moreover, other culture conditions can also be suitablyselected by those skilled in the art, and for example, but not limitedto, the culture temperature can be suitably selected from between35.5-37.5° C., and the culture period can be selected from between 5-20days. For pro-HGFA, the culture period may be set according to thetarget survival rate. The carbon dioxide concentration during culturecan be 5% CO₂ in accordance to the general protocol.

In one embodiment, the method for producing the active HGF of thepresent invention is characterized in that followed by said step, itfurther comprises a step of purifying active HGF. This step may includepurification of pro-HGF that may remain in the preparation comprisingactive HGF.

The purification method that may be used in the present invention is notparticularly limited as long as it enables purification without losingthe physiologic activity of the protein. In particular, it is preferredto use chromatographic purification employing a mixed mode support inthe present invention.

Mixed mode support is also referred to as mixture mode support, and is achromatography support in which ligands of modes with two or more typesof properties are bound into one support. In particular, in the presentinvention, for the purification of active HGF, active HGF can beefficiently purified by chromatographic purification employing a mixedmode support having characteristics of hydrophobicity and ion exchangesupport.

Examples of a “mixed mode support having characteristics ofhydrophobicity and ion exchange support” that may be used in the methodof the present invention can include, but are not limited to, Captoadhere, Capto MMC, HEA HyperCel, PPA HyperCel, MEP HyperCel, TOYOPEARLMX-Trp-650M, and the like.

Chromatographic purification employing said mixed mode support can beperformed by adsorbing active HGF in the column loading solution to saidmixed mode support, and then washing with a buffer to remove impurities,followed by elution. The buffer for removing impurities can be set basedon the pH, electric conductivity, buffer component, salt concentration,or additives that maintain the adsorption between the protein which isthe target for purification and the support while reducing the affinitybetween impurities and the support.

Examples of the column loading solution and buffer used include, but arenot limited to, phosphate salts, citrate salts, acetate salts, succinatesalts, maleate salts, borate salts, Tris (base), HEPES, MES, PIPES,MOPS, TES, or Tricine and the like.

The column loading solution and buffer used can comprise amino acids.Examples of such amino acids can include, but are not limited to,glycine, alanine, arginine, serine, threonine, glutamic acid, asparticacid, histidine, derivatives thereof, and the like.

In the present invention, a column loading solution that has suitable pHand salt concentration for adsorbing active HGF onto said mixed modesupport can be used. Such a pH range is pH 6.0-10.0, more preferably pH7.0-9.0, for example pH 8.0. Moreover, such a salt concentration is0.01-5 M, preferably 0.1-2 M, for example 1 M. The above saltconcentration can be prepared by employing for example 0.001 M-4 Msodium chloride, potassium chloride, calcium chloride, sodium citrate,sodium sulfate, ammonium sulfate, or a combination thereof.

In the present invention, elution of active HGF can be performed byemploying a buffer that will reduce the affinity between said mixed modesupport and active HGF. Such a buffer includes a buffer comprising atleast 0.1 M arginine, more preferably at least 0.3 M arginine, furtherpreferably at least 0.4 M arginine, for example 0.7 M arginine.Moreover, in combination with or instead of arginine, a buffercomprising magnesium ion (Mg²⁺) can also be employed. Alternatively,elution of active HGF may also be performed by a stepwise method thatreduces pH stepwise to elute active HGF.

In one embodiment of the present invention, said purification mayfurther comprise, after purification by a mixture mode supportcomprising an ion exchange group and a hydrophobic interaction group,purification by single or multiple additional chromatographies. Thiswill enable active HGF to be obtained at higher purity. Such achromatographic purification includes, but is not limited to, forexample chromatographic purification that employs a mixed mode support,an anion exchange support, a cation exchange support, a hydrophobicinteraction support, a size exclusion support, a gel filtration support,a reverse phase support, a hydroxyapatite support, a fluoroapatitesupport, a sulfated cellulose support, or a sulfated agarose support andthe like.

Note that the terms used herein are to be employed to describeparticular embodiments and do not intend to limit the invention.

Moreover, the term “comprising” as used herein, unless the contentclearly indicates to be understood otherwise, intends the presence ofthe described items (such as components, steps, elements, and numbers),and does not exclude the presence of other items (such as components,steps, elements, and numbers).

Unless otherwise defined, all terms used herein (including technical andscientific terms) have the same meanings as those broadly recognized bythose skilled in the art of the technology to which the presentinvention belongs. The terms used herein, unless explicitly definedotherwise, are to be construed as having meanings consistent with themeanings herein and in related technical fields, and shall not beconstrued as having idealized or excessively formal meanings.

Terms such as first and second are sometimes employed to express variouselements, and it should be recognized that these elements are not to belimited by these terms. These terms are employed solely for the purposeof discriminating one element from another, and it is for examplepossible to describe a first element as a second element, and similarly,to describe a second element as a first element without departing fromthe scope of the present invention.

The present invention will now be more specifically described byExamples. However, the present invention can be embodied by variousembodiments, and shall not be construed as being limited to the Examplesdescribed herein.

EXAMPLES

The present invention will be specifically described below by showingExamples, but the present invention is not to be limited by theExamples.

Example 1

CHO cells that recombinantly express full length pro-HGFA were thawed inEX-CELL custom design medium (from SAFC) in a T75 flask (from Corning,430421), expansion culture was performed in a 250 mL shaker flask (fromCorning, 431144), and then cultured for 10 days in a 7 L culture tank(from ABLE/Biott, BCP-07) at 121 rpm set at 36.5° C. The survival rateof the cells at Day 10 of culturing was 47.1%. After completion ofculture, cells were removed by centrifugation and microfiltered througha 0.2 μm filter (from Sartorius, 5445307H7-00), and the pro-HGFAsupernatantcollected was stored under refrigeration until use.

50 mL of pro-HGFA culture supernatant obtained similarly as above wasplaced in a 100 mL glass beaker, 5 mL, which is 1/10 volume of thesupernatant, of 10 g/L aqueous solution of dextran sodium sulfate (Mw.500,000) was added, and then pH was adjusted to 5.3 with 2 Mhydrochloric acid. After the pH adjustment, it was subjected tofiltration with a 0.2 μm filter, and then placed in a 250 mL shakerflask. Five percent carbon dioxide was blown in for 60 seconds, and thenreaction was performed at room temperature with stirring speed set at 80rpm for 6 hours. The activation reaction was progressed at around pH5.5. Sampling was performed after 6 hours of reaction, and HGFA activitywas measured with synthetic peptide as the substrate. The syntheticsubstrate H-D-Val-Leu-Arg-pNA2AcOH (from Bachem, L-1885) was dissolvedin 50 mM Tris-HCl-0.15 M sodium chloride-10 mM calcium chloride buffer(pH 7.5) comprising 0.25% BSA, and adjusted to 2 mM. This was applied at100 μL/well in the necessary number of wells in a 96-well plate, and 10μL each of the HGFA culture supernatant which had been subjected toactivation treatment, positive control, and untreated pro-HGFA culturesupernatant were added. As the positive control, HGFA culturesupernatant which had been activated in advance and was confirmed to becapable of sufficiently activating pro-HGF was employed. The plate wasshielded from light with an aluminum foil, and incubated at 37° C. for 1hour. Absorbance was read with a plate reader from TECAN (405 nm), andHGFA activity value was caluculated by subtracting an absorbance ofuntreated pro-HGFA culture supernatant from the original absorbance. Asa result, it was confirmed that the activity value of the HGFA sampleafter activation showed 0.577, which is comparable to the activity valueof the positive control. It is thought that pro-HGFA is activated by theaction of an enzyme derived from the host CHO cell since any enzymes andthe like were not externally added to this reaction solution. Moreover,when 1 M Tris was added to the solution after 7.6 hours of reaction toadjust pH to 7.0 and then the solution was stored under refrigerationfor 2 days to examine the change in HGFA activity value, a large declinein the activity value was not seen with the activity value immediatelyafter neutralization at 0.653, Day 1 of refrigeration at 0.667, and Day2 of refrigeration at 0.679, showing stability for 2 days afteractivation (Table 1).

TABLE 1 HGFA Activity Value After Activation Treatment HGFA ActivitySample A405 Value Measurement 1 Pro-HGFA 0.100 — culture supernatant 6hours after 0.677 0.577 activation Positive-control 0.709 0.609Measurement 2 Pro-HGFA 0.110 — culture supernatant 7.6 hours after 0.8030.693 activation (before neutralization) 7.6 hours after 0.763 0.653activation (after neutralization) Day 1 of 0.777 0.667 refrigeratedstorage after neutralization Day 2 of 0.789 0.679 refrigerated storageafter neutralization Positive-control 0.714 0.604

CHO cells that recombinantly express pro-HGF were thawed in EX-CELLcustom design medium in a T75 flask, expansion culture was performed ina 250 mL shaker flask and a 7 L culture tank, and this was then culturedfor 9 days in a 20 L culture tank at 144 rpm set at 36.5° C. Thesurvival rate at Day 9 of culturing was 90.6%. After filtration toremove cells, 19.14 kg of pro-HGF culture supernatant that had beenmicrofiltered through a 0.2 μm filter (from Sartorius, 5445307H9--00)was charged into a 30 L culture tank. To this, 0.96 kg, which is 1/20volume of the HGF supernatant, of the HGFA culture supernatant that hadbeen activated and returned to pH 7.0 and stored under refrigeration for2 days was added and reacted with stirring at 30 rpm at 25° C. Note thatthe activated HGFA culture supernatant charged was that which had anactivity value comparable to the positive control in HGFA activitymeasurement. Sampling was performed after about 20 hours of reaction,and the activation state of pro-HGF was confirmed with SDS-PAGEemploying 5-20% polyacrylamide gel (from DRC, NXV-271HP). A band ofsingle strand was seen under a non-reductive condition, and under areductive condition after activation the single strand substance haddisappeared and separated into α and β chains, and thus sufficientactivation of pro-HGF was confirmed (FIG. 1).

Example 2

Using design of experiments (DoE), the validity of pro-HGFA activationparameters described in Example 1 which are pH (5.3-5.5) and reactiontemperature (room temperature) was tested. Experiment conditions wereset with central composite design using JMP software (from SASInstitute), and a solution for pro-HGFA activation treatment wasprepared similarly to the method described in Example 1. Note that pHwas adjusted to three conditions of pH 5.0, 5.5, and 6.0 with 2 Mhydrochloric acid. 100 μl each were placed in 1.5 mL tubes and reactedby still standing at 20° C., 28.5° C. and 37° C. Sampling was performedafter 3, 6, 9, and 15 hours of reaction, and HGFA activity measured withsynthetic peptide as the substrate. HGFA activity value is obtained bysubtracting the value (A405) of untreated pro-HGFA culture supernatant.A response surface plot was created by statistical analysis from theHGFA activity values obtained from a total of 27 conditions, and therange having an activity value of 0.4 or more was shown in white. Fromthis result, it was found that the condition that gives the highest HGFAactivity value is pH 5.4 and a reaction temperature of 26.1° C., andthat HGFA activity value can be obtained in a wide range (FIG. 2).

Example 3

2 mL of multimodal anion exchanger Capto Adhere (from GE Healthcare,28-4058-44) was equilibrated in advance with 20 mM Tris-hydrochloridebuffer (pH 8.0) comprising 2 M sodium chloride. To 32 mL of culturesupernatant comprising active HGF, sodium chloride was added to obtain 1M. This culture supernatant was loaded onto the column at a flow rate of2 mL/min and the flow-through solution was collected. After loading wascomplete, 20 mM Tris-hydrochloride buffer (pH 8.0) comprising 2 M sodiumchloridewas flowed at an amount corresponding to 3 times of the columnvolume to wash, and the eluate was collected. After washing wascomplete, 20 mM Tris-hydrochloride buffer (pH 8.0) comprising 0.25 Marginine was flowed at an amount corresponding to 3 times of the columnvolume to wash and the eluate was collected. Next, an operation to flow20 mM Tris-hydrochloride buffer (pH 8.0) comprising 0.7 M arginine at anamount corresponding to 1 column volume to collect the eluate wasrepeated 5 times. Finally, 20 mM Tris-hydrochloride buffer (pH 8.0)comprising 1.0 M arginine was flowed at an amount corresponding to 3times of the column volume to collect the eluate. FIG. 3 shows theresult of performing SDS-PAGE under a non-reductive condition with thesolutions collected in this process. The gel for SDS-PAGE employed wasXV-PANTERA (NXV-271HP) from DRC, and the molecular weight markeremployed was Precision Plus Protein All Blue Standards (161-0373) fromBIORAD. The samples were subjected to SDS-PAGE analysis after performing10 minutes of heat treatment in Laemmli's sample buffer at 60° C.Electrophoresis was performed under a constant voltage of 150 V, and thegel was stained when the electrophoresis was complete with PAGE Blue83from COSMO BIO to confirm the separated proteins. When comparing thecolumn loading solution and the flow-through solution, the HGF band ofmolecular weight of around 75,000 was decreased in the flow-throughsolution, showing that it was adsorbed onto the support. HGF was noteluted by flowing through 20 mM Tris-hydrochloride buffer (pH 8.0)comprising 2 M sodium chloride. A component comprising much impurity waseluted in subsequent washing with 20 mM Tris-hydrochloride buffer (pH8.0) comprising 0.25 M arginine. Active HGF was then eluted by flowingthrough 20 mM Tris-hydrochloride buffer (pH 8.0) comprising 0.7 Marginine.

Example 4

1 mL of multimodal anion exchanger Capto Adhere (from GE Healthcare,28-4058-44) was equilibrated in advance with 20 mM Tris-hydrochloridebuffer (pH 8.0) comprising 2 M sodium chloride. To 8 mL of culturesupernatant comprising active HGF was added an equal amount of 20 mMTris-hydrochloride buffer (pH 8.0) comprising 2 M sodium chloride toobtain 1 M. This culture supernatant was loaded onto the column and theflow-through solution was collected. After loading was complete, 20 mMTris-hydrochloride buffer (pH 8.0) comprising 2 M sodium chloride wasflowed at an amount corresponding to 3 times of the column volume towash, and the eluate was collected. An amount corresponding to 5 timesof the column volume of 20 mM Tris-hydrochloride buffer (pH8.0)comprising 1 M arginine was flowed, and the eluate was collected.FIG. 4 shows the result of performing SDS-PAGE under a non-reductivecondition with the solutions collected in this process. The gel forSDS-PAGE employed was XV-PANTERA (NXV-271HP) from DRC, and the molecularweight marker employed was Precision Plus Protein All Blue Standards(161-0373) from BIORAD. The samples were subjected to SDS-PAGE analysisafter performing 10 minutes of heat treatment in Laemmli's sample bufferat 60° C. Electrophoresis was performed under a constant voltage of 150V, and the gel was stained when the electrophoresis was complete withPAGE Blue83 from COSMO BIO to confirm the separated proteins. Whencomparing the column loading solution and the flow-through solution, HGFband was decreased in the flow-through solution, showing that it wasadsorbed onto the support. HGF was not eluted by flowing through 20 mMTris-hydrochloride buffer (pH 8.0) comprising 2 M sodium chloride.Active HGF was eluted with the subsequent elution with 20 mMTris-hydrochloride buffer (pH 8.0) comprising 1 M arginine.

Example 5

To the culture supernatant comprising active HGF obtained in the methodof Example 1 was added an equal amount of 40 mM Tris-hydrochloridebuffer (pH 8.0) comprising 2 M sodium chloride, and then the pH wasadjusted to 8.0. The above solution was loaded onto a Capto adhere (GEHealthcare, 17-5444-05) column equilibrated with 20 mMTris-hydrochloride buffer (pH 8.0) comprising 2 M sodium chloride, andafter loading was complete, washing with the buffer employed forequilibration was performed. The column was washed with 20 mMTris-hydrochloride buffer (pH 8.0) comprising 0.25 M argininehydrochloric acid, after which it was eluted with 20 mMTris-hydrochloride buffer (pH 8.0) comprising 0.7 M argininehydrochloric acid, and the fraction comprising HGF was collected.

The Capto adhere purification fraction was pooled, the solution diluted7 times with 20 mM Tris-hydrochloride buffer (pH 7.5) comprising 0.012%polysorbate 80 was loaded onto a Capto Q (GE Healthcare, 17-5316-05)column equilibrated with 20 mM Tris-hydrochloride buffer (pH 7.5)comprising 0.012% polysorbate 80, and after loading was complete,washing with the buffer employed for equilibration was performed. Thecolumn flow-through solution and the wash solution were pooled as theCapto Q purification fraction.

The Capto Q purification fraction was loaded onto a UNOsphere S (Bio-Rad156-0117) column equilibrated with 20 mM phosphate buffer (pH 7.5), andafter loading was complete, this was washed with the buffer employed forequilibration. After completion of washing with the same solution, thiswas washed with 20 mM phosphate buffer (pH 7.5) comprising 0.4 M sodiumchloride, and then the adsorbed HGF was eluted with 20 mM phosphatebuffer (pH 7.5) comprising 0.6 M sodium chloride as the UNOsphere Spurification fraction.

To the UNOsphere S purification fraction was added 20 mM phosphatebuffer (pH 7.5) comprising 5 M sodium chloride to adjust the sodiumchloride concentration of the solution to 3.3 M and the pH to 7.5.Phenyl Sepharose HP (GE Healthcare, 17-1082-04 column) was equilibratedwith 20 mM phosphate buffer (pH 7.5) comprising 3.3 M sodium chloride,and then the above HGF solution was loaded. After loading was complete,the column was washed with the buffer employed for equilibration. Theadsorbed HGF was eluted by a linear gradient of the equilibration buffer(A) and 20 mM phosphate buffer (pH 7.5) (B) (from 30 to 100% of B).

Example 6

For the culture supernatant comprising unactivated pro-HGF solution towhich active HGFA was not added, Capto adherese purification, CaptoQpurification, UNOsphereS purification, and UF concentration bufferexchange were carried out similarly to Example 5. Non-reductive andreductive SDS-PAGE results of samples obtained in each step shown inFIG. 5 show that the unactivated pro-HGF is also purified in the presentpurification process.

Example 7

For the active HGF obtained in Example 5, cell proliferation activity inthe presence of TGFβ-1 was measured. Using mink lung epithelial cell My1 Lu (cell No.: JCRB9128), active HGF was added to cells of which thegrowth was inhibited in the presence of Transforming Growth Factor β-1(TGFβ-1), and the active HGF proliferation activity thereof based on theantagonistic action on TGFβ-1 activity was detected to measure the titer(Journal of Immunological Methods, 258, 1-11, 2001).

In each well of a 96-well plate, 50 μL of TGFβP-1 (4 ng/mL), 50 μL eachof International HGF reference standard (NIBSC code: 96/564) or HGF (0,4, 8, 16, 32, 64, 128, 256, 512, and 1024 ng/mL), and 100 μL of minklung epithelium cell suspension (1×10⁵ cells/mL) were added and culturedat 37° C., 5% CO₂ concentration for 3 days, and then viable cells werestained by Cell counting kit (DOJINDO LABORATORIES, Cat No. 343-07623).Using a microplate reader, sigmoid curves were obtained for each ofInternational HGF reference standard and HGF from absorbance at 450 nm(FIG. 6). EC50 of International HGF reference standard and HGF was 13.4and 15.4 ng/mL, respectively, and HGF obtained with the above productionmethod had activity equivalent to that of the International HGFreference standard.

Sequence Listing

ESAP1601F sequence listing.txt

1-13. (canceled)
 14. A polypeptide selected from the group consistingof: (i) the amino acid sequence shown in SEQ ID NO:1; (ii) the aminoacid sequence shown in SEQ ID NO:1 which has substitution, deletion,insertion, and/or addition of 1 to 10 amino acid residues, and has HGFactivity similar to or more than the polypeptide having the amino acidsequence shown in SEQ ID NO:1 or which is activated to have theactivity; and (iii) the amino acid sequence showing an identity of 95%or higher to the amino acid sequence of SEQ ID NO:1 and having HGFactivity similar to or more than the polypeptide having the amino acidsequence shown in SEQ ID NO:1 or which is activated to have theactivity.
 15. A polypeptide selected from the group consisting of: (i)the amino acid sequence shown in SEQ ID NO:2, (ii) the amino acidsequence shown in SEQ ID NO:2 which has substitution, deletion,insertion, and/or addition of 1 to 10 amino acid residues, and havingHGFA activity similar to or more than the polypeptide having the aminoacid sequence shown in SEQ ID NO:2 or which is activated to have theactivity, and (iii) the amino acid sequence showing an identity of 95%or higher to the amino acid sequence of SEQ ID NO:2 and having HGFAactivity similar to or more than the polypeptide having the amino acidsequence shown in SEQ ID NO:2 or which is activated to have theactivity.